Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that participate in host immunity to cancer. Despite the recognition that iNKT cells promote anti-tumor immune responses, many questions remain regarding how these cells recognize and respond to tumor cells. This lack of information impedes the development of novel and potentially more effective iNKT cell-based immunotherapies for cancer. My studies reveal that primary murine and human iNKT cells exhibit robust T cell receptor (TCR)-induced cytolytic activity against tumor cells in vitro and in vivo. In an attempt to elucidate the signalig mechanisms that regulate TCR-induced iNKT cell responses, I recently observed that immunoglobulin superfamily receptors, CD2 and 2B4 differentially regulate murine iNKT cell cytotoxicity. Specifically, Cd2-/- iNKTs completely failed to kill tumor targets while 2b4-/- iNKTs exhibited significantly enhanced cytolysis. Based upon these findings, I hypothesize that CD2 is a positive and 2B4 a negative regulator of TCR-induced iNKT cell functions. In this K22 proposal, I will build upon these new and exciting observations by further exploring the roles of CD2 and 2B4 in iNKT cell-mediated tumor clearance in vivo (Aim 1a) and iNKT cell immuno-stimulatory functions (cytokine production and activation of other immune cells) in vitro and in vivo (Aim 1b). Additionally, I will also examine the roles of these receptors in human iNKT cell activation, proliferation, cytokine production and anti- tumor responses in vitro (Aim 1c). To dissect the mechanistic basis by which these molecules regulate iNKT cell cytotoxicity, I will assess how disruption of CD2 or 2B4 signaling affects iNKT cell immunological synapse formation and maturation (Aim 2a). CD2 and 2B4 both bind to their ligand, CD48 but with different affinities. Furthermore, CD2 and 2B4 have different cytoplasmic tails, which are likely t confer distinct signaling functions. Therefore, I will delineate whether CD2 and 2B4 exert opposing roles by inducing distinct intracellular signals (Aim 2b) and/or by competing with one another for CD48 binding (Aim 2c). As CD1d (the ligand for the invariant TCR) and CD48 (the ligand for CD2 and 2B4) are both widely expressed on hematopoietic tumors, further elucidation of the mechanisms by which CD2 and 2B4 regulate TCR-induced iNKT cell anti- tumor activity is of significant scientific and clinical importance. These studies will facilitate a better understanding of iNKT cell biology and provide insights into how the anti-tumor activities of iNKT cells can be harnessed in a therapeutically relevant manner. To enable the successful completion of these studies and facilitate my scientific and career development, I have established critical collaborations and a scientific advisory committee that includes Drs. Stephan Grupp, Jordan Orange and Taku Kambayashi, all experts in their respective fields of investigation. I will take advantage of the intellectual strength and academic track record of my mentor and scientific advisory committee members, and the robust availability of expertise, facilities, and resources offered at The Children's Hospital of Philadelphia and the University of Pennsylvania to accomplish the training program outlined in this proposal.